PROJECT SUMMARY Individuals living with cystic fibrosis (CF) combat devastating, chronic microbial infections of the airways. Though CF patients are usually colonized by otherwise innocent bacteria and fungi?many of which derive from soil environments?the compromised CF immune system is unable to clear these opportunists, and the ensuing struggle between host and microbes eventually leads to failure of the pulmonary system. Conventional antibiotics are not very effective. One widely held view for why this is the case is that the resident microbiota are growing slowly and their membranes are insufficiently charged to take up commonly-used antibiotics such as tobramycin. Accordingly, if we seek new ways to treat CF lung infections, we must better understand how microbes thrive in this habitat. Different lines of evidence indicate that oxygen is limiting in the CF airways at the microscale relevant to opportunistic pathogens. Though carbon sources are replete in the mucus-filled airways, in the absence of oxygen, the bacteria and fungi that come to dominant this habitat must employ alternative energy generation strategies to aerobic respiration. How do they do this? Energy flow is the driving organizer of any microbial community, including those found in the CF airways. It is well established that the nitric oxide (NO) generated by the immune system in the CF patients is lower than that made by healthy individuals. Indeed, epithelial cells are severely compromised in NO production, yet neutrophils that flood the CF airways can still generate NO; this NO is insufficient to kill the microbes, but has the potential to transform into an energy source for those capable of denitrification. Accordingly, we seek to test the hypothesis that microbial energetic hijacking of the CF immune response via denitrification pathways selects for specific pathogens during lung function decline. To test this hypothesis, we plan to leverage powerful isotopic tools from the Earth sciences that permit the sources of metabolites in complex environments, such as the CF airways, to be determined atom by atom, providing a non-invasive, forensic molecular fingerprint. In particular, we will focus on interpreting N2O, a measurable product of the denitrification pathway in CF sputum and breath gas. In addition to approaches employing bacterial physiology, genetics, and in situ imaging of host-microbe interactions, we will use these isotopic tools to gain insight into how the NO that is reduced to N2O in the CF airways may favor the fitness of particular microbial community members. To do this, we propose three specific aims: Aim 1 will identify the conditions and pathways leading to N2O production by common CF bacteria and fungi, Aim 2 will utilize advanced isotopic analyses to dissect the N2O produced by these microbes to forensically infer its source, and Aim 3 will apply these insights to a pilot study of adult CF patients to determine whether particular types of denitrifiers are favored as lung function declines. Attainment of these objectives will provide the critical knowledge needed to guide early translational approaches for treating infections of the CF airways, as well as establish tools that can be applied more broadly to other studies of dysbiosis.